Methods of treating CXCR4-expressing cancers

ABSTRACT

Some embodiments of this invention include methods for treating disease and methods for administering a compound of Formula (I). In some aspects of the invention, diseases can be treated by administration of compositions comprising a compound of Formula (I). Pharmaceutical compositions of some embodiments of the present invention comprise a compound of Formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/639,864 with a 371 date of Oct. 19, 2012, which isincorporated by reference in its entirety, which is a National StageEntry of International Application No. PCT/US2011/031654 filed Apr. 8,2011, which is incorporated by reference in its entirety, which claimsbenefit to U.S. Provisional Application No. 61/322,485, filed Apr. 9,2010, which is incorporated by reference in its entirety.

GOVERNMENT RIGHTS

The invention described was made with government support under GrantNumber DAMD17-02-1-0446 awarded by the U.S. Department of Defense. Thegovernment has certain rights in this invention.

BACKGROUND

CXCR4 is a class A G-protein coupled receptor (GPCR) that can bindstromal-derived factor 1 (SDF-1 also known as CXCL12), a CXC chemokine.Chemokines are members of a gene family of cytokines that can promoteinflammatory and immunological responses by effecting the recruitment ofappropriate leukocyte populations. Although chemokines have beencharacterized as promoting directed migration of leukocytes, they canalso have roles outside the hematopoietic compartment. CXCR4 can beexpressed constitutively in normal tissue.

Class A GPCRs have been characterized structurally by seven membranespanning helical domains, an extracellular amino terminus, and a carboxyterminus on the intracellular side of the membrane. The seventransmembrane (TM) domains appear to be joined by three extracellular(ECL) and three intracellular (ICL) loops. Some crystal structures ofCXCR4 appear to be reported in Wu et al., Science, Vol. 330, pp.1066-1071 (2010).

CXCR4 is a chemokine receptor and a natural ligand for CXCR4 is thechemokine CXCL12. It appears that CXCR4 can be expressed on the surfaceof breast cancer cells. And in some instances, CXCR4 can play a role inboth angiogenesis and metastasis in several tumor types, including, butnot limited to, basal cell carcinoma, thyroid cancer, squamous cellcarcinoma, neuroblastoma, ovarian, melanoma, renal cell carcinoma,hepatocellular carcinoma, breast, colon, lung, pancreatic, and prostatecancers. CXCR4 expression by the tumor cells appears to be involved intumor cell migration and in homing of the neoplastic cells to siteswhere non-malignant stromal cells express CXCL12.

Accordingly, there exists a further need (1) for treatment of disease(e.g., cancer) with a composition that may target CXCR4 or a G-protein,(2) for modulation of activity of CXCR4 with a composition, and (3) forproviding pharmaceutical compositions that may treat diseases related toCXCR4 or G-proteins. Some embodiments of the present invention mayaddress one or more of these needs.

SUMMARY

Some embodiments of the invention include methods for treating a diseasein an animal comprising administering a compound of Formula (I) to theanimal. Similarly, in some embodiments, a compound can be used intreating disease in an animal. The animal can be a mammal, such as ahuman or a mouse. In some instances, this method further comprisesidentifying an animal with the disease. The disease can be cancer orinclude a cancerous tumor. In some aspects, the treating results in adecrease in the size of the cancerous tumor, a decrease in the number ofcancerous tumors, or both. In some embodiments, the disease is cancer,systemic lupus erythematosus, HIV, Epstein barr virus, coronary arterydisease in type II diabetes melitus, chronic rhinositis, carotid arterystenosis, choroidal neovascularization, bladder hyperreflexia,nephrosclerosis, basal cell carcinoma, thyroid cancer, squamous cellcarcinoma, neuroblastoma, ovarian cancer, melanoma, renal cellcarcinoma, hepatocellular carcinoma, breast cancer, colon cancer, lungcancer, pancreatic cancer, prostate cancers, chronic lymphocyticleukemia (CLL), acute lymphoblastic leukemia, rhabdomyosarcoma,Glioblastoma multiforme, meningioma, bladder cancer, gastric cancer,Glioma, oral cancer, nasopharyngeal carcinoma, kidney cancer, rectalcancer, stomach cancer, uterine cancer, or leukemias. In certainembodiments, the treatment includes treatment of angiogenesis ormetastasis. Sometimes the treatment includes modulation of at least oneof (1) actin modification, (2) pseudopodia formation, (3) tumor cellmigration, or (4) homing of the neoplastic cells.

Some embodiments of the method of treating include administering by anoral route or by a parenteral route. In still other embodiments,administering is part of an adjuvant treatment.

Some other embodiments of the method of treating include a compound ofFormula (I) that inhibits chemotaxis, inhibits intracellular calciummobilization, modulates activity of a G-protein, or modulates activityof CXCR4. In some instances, the compound of Formula (I) is selectedfrom the group consisting of I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8,I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20,I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32,I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44,I-45, and I-46 (i.e., selected from the group consisting of I-1 toI-46), or can be selected from the group consisting of I-2, I-4, I-7,T-9, I-25, I-28, I-36, I-38, and I-43.

Still other embodiments of the invention include a method ofadministering a composition to a cell comprising administering thecomposition comprising at least one compound of Formula (I) to the cell.The cell can be an animal cell such as Lewis lung carcinoma cells,B16F10 melanoma cells, TC-1 cervical carcinoma cells, HS27 cells, MCF7cells, MDA-MB-231 cells, A549 cells, THP-1 cells, 300.19 cells, CHOcells, mouse cells, or African green monkey cells. The cell can be amammalian cell, such as a human cell or a mouse cell. The cell can be atransfected cell. The cell can be part of an organ or from amulticellular organism.

Some embodiments of the method of administering include a compound ofFormula (I) that inhibits chemotaxis of the cell, inhibits intracellularcalcium mobilization in the cell, modulates activity of a G-protein, ormodulates activity of CXCR4. The compound can be selected from the groupconsisting of I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11,I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23,I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35,I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, and I-46(i.e., selected from the group consisting of I-1 to I-46). Or thecompound can be selected from I-2, I-4, I-7, I-9, I-25, I-28, I-36,I-38, or I-43.

In some instances, the method of administering modulates at least one of(1) actin modification, (2) pseudopodia formation, (3) tumor cellmigration, or (4) homing of neoplastic cells.

Still other embodiments of the invention include compositions comprisinga compound of Formula (I), wherein the composition is a pharmaceuticalcomposition. In some instances, the compound of Formula (I) can, inhibitchemotaxis, inhibit intracellular calcium mobilization, modulateactivity of a G-protein, or modulate activity of CXCR4. The compound canbe selected from the group consisting of I-1, I-2, I-3, I-4, I-5, I-6,I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18,I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30,I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42,I-43, I-44, I-45, and I-46 (i.e., selected from the group consisting ofI-1 to I-46), or can be selected from the group consisting of I-2, I-4,I-7, I-9, I-25, I-28, I-36, I-38, and I-43.

In some embodiments, the compound of Formula (I) is present in atherapeutically effective amount, such as a therapeutically effectiveamount to treat cancer or a therapeutically effective amount to treat orprevent metastasis. In some aspects, the compound of Formula (I) ispresent in a therapeutically effective amount to decrease the size of acancerous tumor, to decrease the number of cancerous tumors, or both.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of thepresent invention can be best understood when read in conjunction withthe following drawings:

FIG. 1A shows the effect of the identified compounds on chemotaxis.

FIG. 1B shows the selectivity of chemokine receptors.

FIG. 1C shows inverse agonist activity against CXCR4.

FIG. 2A shows results from the chemotaxis assay method.

FIG. 2B shows the chemotaxis IC50 of I-38 and I-43.

FIG. 3A shows the effect of compounds on intracellular calciummobilization.

FIG. 3B shows IC50 of intracellular calcium mobilization for severalcompounds.

FIG. 4 shows cytotoxicity of some compounds.

FIG. 5A shows the chemotaxis index of some compounds.

FIG. 5B shows the chemotaxis IC50 of some compounds.

FIG. 6A shows intracellular calcium mobilization upon treatment of theindicated compounds.

FIG. 6B shows the IC50 of intracellular calcium mobilization.

FIG. 7A shows cytotoxicity using propidium iodide of some compounds.

FIG. 7B shows cytotoxicity IC50 values of some compounds.

FIG. 8A shows inhibition of chemotaxis in cells expressing CXCR4 and incells expressing BLT1. “Chemokine” is CXCL12 for cells expressing CXCR4and LTB4 for cells expressing BLT1.

FIG. 8B shows the chemotaxis index in cells expressing CXCR4 and incells expressing BLT1. “Chemokine” is CXCL12 for cells expressing CXCR4and LTB4 for cells expressing BLT1.

FIG. 9 shows the chemotaxis IC50 in cells expressing CXCR4 and in cellsexpressing BLT1.

FIG. 10A shows inhibition of intracellular calcium mobilization in cellsexpressing CXCR4 and in cells expressing BLT1. “Chemokine” is CXCL12 forcells expressing CXCR4 and LTB4 for cells expressing BLT1.

FIG. 10B shows the intracellular calcium mobilization in cellsexpressing CXCR4 and in cells expressing BLT1. “Chemokine” is CXCL12 forcells expressing CXCR4 and LTB4 for cells expressing BLT1.

FIG. 11 shows the IC50 measurements for intracellular calciummobilization in cells expressing CXCR4 and in cells expressing BLT1.

FIG. 12A shows proliferation inhibition in normal cell line, HS27 cells.

FIG. 12B shows proliferation inhibition in MCF7 cells.

FIG. 12C shows proliferation inhibition in MDA-MB-231 cells.

FIG. 12D shows proliferation inhibition in A549 cells.

FIG. 13A shows expression of CXCR4 on TC-1 cells; FIGS. 13B and 13C showtreatment of mice to assess reduction of tumor incidence and of tumorburden, respectively.

DETAILED DESCRIPTION

Compounds I-1 to I-62, below, comprise the genus of Formula (I).

Compound I-1 is1-[(1,3-benzodioxol-5-ylmethyl)amino]-3-(4-nitrophenoxy)propan-2-ol,identified by targeting intracellular loops of CXCR4.

Compound I-2 is 1-(2-naphthyl)-5-(2-pyrrolidin-1-ylvinyl)tetrazole,identified using the target Gαi.

Compound I-3 is2-(5-(4-pentylphenyl)-4H-1,2,4-triazol-3-ylthio)-1-(4-nitrophenyl)ethanone,identified by targeting intracellular loops of CXCR4.

Compound I-4 isN-[2-(4-ethylphenoxy)ethyl]-5-(3-methylpiperazin-1-yl)-2-nitro-aniline,identified using the target Gαi.

Compound I-5 is1-(4-chlorophenoxy)-3-[2-imino-3-[2-(1-piperidyl)ethyl]benzoimidazol-1-yl]-propan-2-ol,identified using the target Gαi.

Compound I-6 isN-(3-dimethylaminopropyl)-N-[(4-isopropenyl-1-cyclohexenyl)methyl]-N′,N′-dimethyl-propane-1,3-diamine,identified using the target Gαi.

Compound I-7 isN-((1-(2-(o-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)methyl)-4-methoxybenzenamine,identified by targeting intracellular loops of CXCR4.

Compound I-8 is4-ethoxy-N-((1-(2-phenoxyethyl)-1H-benzo[d]imidazol-2-yl)methyl)benzenamine,identified by targeting intracellular loops of CXCR4.

Compound I-9 isN-(5-(6-methoxyquinolin-8-ylamino)pentan-2-yl)benzo[g]quinolin-4-amine,identified by targeting intracellular loops of CXCR4.

Compound I-10 isN-[(1-methylbenzoimidazol-2-yl)methyl]-1-propyl-benzoimidazol-5-amine,identified using the target Gαi.

Compound I-11 is1-[[4-[[4-[(2,5-dioxopyrrolidin-1-yl)methylamino]phenyl]methyl]phenyl]aminomethyl]pyrrolidine-2,5-dione,identified using the target Gαi.

Compound I-12 is 2-[(3-cyano-5,7-dimethyl-2-quinolyl)amino]ethyl2-methoxybenzoate, identified by targeting intracellular loops of CXCR4.

Compound I-13 isN-[2-[(3-cyano-5,7-dimethyl-2-quinolyl)amino]ethyl]propanamide,identified by targeting intracellular loops of CXCR4.

Compound I-14 is1-[(3,4,5-trimethoxyphenyl)methyl]-spiro[piperidine-4,4′(5′H)-pyrrolo[1,2-a]quinoxaline],identified by targeting intracellular loops of CXCR4.

Compound I-15 is5-[3-[(4-ethoxyphenyl)amino]-2-hydroxypropyl]-1,5-dihydro-3-methyl-1-oxo-pyrido[1,2-a]benzimidazole-4-carbonitrile,identified by targeting intracellular loops of CXCR4.

Compound I-16 is 1-carbazol-9-yl-3-(4-ethoxyphenyl)amino-propan-2-ol,identified by targeting intracellular loops of CXCR4.

Compound I-17 is2-[3-(3-dimethylaminopropyl)-2-imino-benzoimidazol-1-yl]-1-phenyl-ethanol,identified by targeting intracellular loops of CXCR4.

Compound I-18 is1-[[(2,3-dihydro-1,4-benzodioxin-2-yl)methyl]amino]-3-(4-nitrophenoxy)-2-propanol,identified by targeting intracellular loops of CXCR4.

Compound I-19 is1-methyl-2-(1-piperidylmethyl)-N-(2-pyridylmethyl)benzoimidazol-5-amine,identified by targeting intracellular loops of CXCR4.

Compound I-20 is2-(1,3-benzodioxol-5-yl)-4-[(4-methylphenyl)methyl]imidazo[1,2-a]benzimidazole,identified by targeting intracellular loops of CXCR4.

Compound I-21 is1-[[1-(2,3-dihydro-1,4-benzodioxin-2-yl)ethyl]amino]-3-(4-nitrophenoxy)-2-propanol,identified by targeting intracellular loops of CXCR4.

Compound I-22 isN-[(1-benzylbenzoimidazol-2-yl)methyl]-1,2-dimethyl-benzoimidazol-5-amine,identified using the target Gαi.

Compound I-23 is5-(2-morpholinoethylamino)-2-[(4-phenylphenoxy)methyl]oxazole-4-carbonitrile,identified by targeting intracellular loops of CXCR4.

Compound I-24 isN-(3,4-dimethylisoxazol-5-yl)-4-(3-phenylsulfonylaminoquinoxalin-2-yl)amino-benzenesulfonamide,identified by targeting intracellular loops of CXCR4.

Compound I-25 isN-[3-(3-dimethylaminophenyl)aminoquinoxalin-2-yl]-4-nitro-benzenesulfonamide,identified by targeting intracellular loops of CXCR4.

Compound I-26 is1-[1-(4-fluorophenyl)-2,5-dimethyl-pyrrol-3-yl]-2-(2-furylmethylamino)ethanone,identified using the target Gαi.

Compound I-27 isethyl-2-(2-benzothiazol-2-ylsulfanyl-2-phenyl-acetyl)amino-6-methyl-4,5,6,7-tetrahydrobenzothiophene-3-carboxylate,identified by targeting intracellular loops of CXCR4.

Compound I-28 is4-[[2-(3,4-dimethylphenyl)-1,3-dioxo-4-isoquinolylidene]methylamino]-N-(4-hydroxyphenyl)-benzenesulfonamide,identified by targeting intracellular loops of CXCR4.

Compound I-29 is[2-[1-(1,5-dimethyl-3-oxo-2-phenyl-pyrazol-4-yl)-2,5-dimethyl-pyrrol-3-yl]-2-oxo-ethyl]2-(3,5-dimethyl-4-nitro-pyrazol-1-yl)acetate,identified by targeting intracellular loops of CXCR4.

Compound I-30 is1-[3-[3-[2-(3,4-dimethoxyphenyl)ethylamino]-2-hydroxy-propoxy]phenyl]ethanone,identified by targeting intracellular loops of CXCR4.

Compound I-31 isN-(3,4-dimethylphenyl)-2-[[5-(4-methoxyphenyl)amino-1,3,4-thiadiazol-2-yl]sulfanyl]acetamide,identified by targeting intracellular loops of CXCR4.

Compound I-32 is[2-(4-ethoxycarbonyl-3,5-dimethyl-1H-pyrrol-2-yl)-2-oxo-ethyl]5-methyl-3-phenyl-isoxazole-4-carboxylate,identified by targeting intracellular loops of CXCR4.

Compound I-33 is2-(2-chlorophenyl)-N′-(4-methyl-1-cyclohexenyl)-quinoline-4-carbohydrazide,identified by targeting intracellular loops of CXCR4.

Compound I-34 is 1-(1-naphthylcarbamoyl)ethyl2-(3,5-dimethyl-4-nitro-pyrazol-1-yl)acetate, identified by targetingintracellular loops of CXCR4.

Compound I-35 is (4-methoxycarbonylphenyl)methylcarbamoylmethyl5-methyl-3-phenyl-isoxazole-4-carboxylate, identified by targetingintracellular loops of CXCR4.

Compound I-36 isN-[3-(9-ethylcarbazol-2-yl)aminoquinoxalin-2-yl]-4-methyl-benzenesulfonamide,identified by targeting intracellular loops of CXCR4.

Compound I-37 is2-(3,4-dimethylphenyl)amino-N-[5-[[3-(trifluoromethyl)phenyl]methyl]thiazol-2-yl]-acetamide,identified using the target Gαi.

Compound I-38 is acetic acid,2-[[(phenylmethylene)amino]oxy]-,2-[1-[[bis(phenylmethyl)amino]methyl]-1,2-dihydro-2-oxo-3H-indol-3-ylidene]hydrazide,identified by targeting extracellular loops of CXCR4.

Compound I-39 is methyl4-(2,3-di(pyridin-2-yl)quinoxaline-6-carboxamido)benzoate, identified bytargeting extracellular loops of CXCR4.

Compound I-40 is1-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-3-((E)-3-phenylacryloyl)thiourea,identified by targeting extracellular loops of CXCR4.

Compound I-41 isN-(4-acetylphenyl)-2,3-di(pyridin-2-yl)quinoxaline-6-carboxamide,identified by targeting extracellular loops of CXCR4.

Compound I-42 isN-(4-(3-((pyridin-3-yl)methylamino)-2,5-dioxopyrrolidin-1-yl)phenyl)acetamide,identified by targeting extracellular loops of CXCR4.

Compound I-43 is(3E,5Z)-1-methyl-3,5-bis((pyridin-3-yl)methylene)piperidin-4-one,identified by targeting extracellular loops of CXCR4.

Compound I-44 is(E)-3-amino-N′-cyano-4-(4-methoxyphenyl)-6-phenylbenzo[b]thiophene-2-carboxamidine,identified by targeting extracellular loops of CXCR4.

Compound I-45 is benzenesulfonamide,4-[[5,7-bis(trifluoromethyl)-1,8-naphthyridin-2-yl]oxy]-N-(3-pyridinylmethyl)-,identified by targeting extracellular loops of CXCR4

Compound I-46 is N,N′-1,10-phenanthroline-2,9-diylbis-benzamide,identified by targeting extracellular loops of CXCR4.

Compound T-47 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)-2-(benzo[d]oxazol-2-ylthio)acetohydrazide:

Compound T-48 is(Z)—N′-(1-((dibenzylamino)methyl)-5-bromo-2-oxoindolin-3-ylidene)-2-(3,4-dimethoxyphenyl)acetohydrazide

Compound I-49 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)benzohydrazide.

Compound I-50 is(Z)—N′-(1-((dibenzylamino)methyl)-5-bromo-2-oxoindolin-3-ylidene)benzohydrazide.

Compound I-51 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)isonicotinohydrazide.

Compound I-52 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)nicotinohydrazide.

Compound I-53 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)-2-phenoxyacetohydrazide

Compound I-54 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)-2-phenylacetohydrazide.

Compound I-55 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)thiophene-2-carbohydrazide.

Compound I-56 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)furan-2-carbohydrazide.

Compound I-57 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)-3-methylbenzohydrazide.

Compound I-58 is(Z)—N′-(1-((dibenzylamino)methyl)-2-oxoindolin-3-ylidene)-4-chlorobenzohydrazide.

Compound I-59 is2-nitro-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)benzamide.

Compound I-60 isN¹-(3-((4-aminophenylamino)methyl)benzyl)benzene-1,4-diamine.

Compound I-61 is the following structure.

Compound I-62 is2-methyl-3-((4-((2-methyl-3H-indol-3-yl)(pyridin-2-yl)methyl)piperazin-1-yl)(pyridin-2-yl)methyl)-1H-indole.

Although some of the compounds have been identified by a particularvirtual screening interface, any of the compounds of Formula (I) couldinteract with one or more parts of the G-protein that may result inmodulation of activity, including but not limited to, inhibition bydirect interaction with one or more interfaces, stimulation by directinteraction with one or more interfaces, or allosteric modulation ofactivity. For example, the compounds of Formula (I) may, but are notrequired to, affect activity in one or more of the following ways (a)modulate signaling in the pertussis toxin-sensitive Gi pathway, (b)modulate dissociation of the Gαi from the trimeric G-proteins, (c)modulate activation of Gβγ by one or more phosphorylation pathways,including, but not limited to, ERK1/2, MAPK, and GSK 3α/β, (d) modulateactivation of the Gβγ subunit which may result in one or more signalingevents, including, but not limited to signaling by the Phospholipase Cpathway to enhance calcium release and signaling by thePhosphoinositide-3-kinase pathway to activate AKT, (e) modulateintracellular cAMP levels, (f) modulate activation of cAMP-dependantsignaling pathways, including, but not limited to PKa or CREB, (g)modulation of cell migration, or (h) modulate one or more changes incell phenotype, including, but not limited to cell adhesion (e.g., bymodulating binding between integrin couples, such as VLA-4 and VCAM-1),modulating invasiveness by increasing AKT mediated MMP9 expression, andmodulating facilitation of cytoskeletal rearrangement by stimulatingactin polymerization by formation of F-actin.

In some embodiments, a compound of Formula (I) can induce a reduction inchemotaxis (e.g., relative to optimized CXCL12) of a CXCR4-expressingcell of no more than about 10%, no more than about 25%, no more thanabout 50%, no more than about 75%, or no more than about 90%. Somecompounds of Formula (I) can have selectivity for CXCR4 chemotaxisinhibition. For example, the ratio of chemotaxis inhibition for cellsexpressing non-CXCR4 inducing chemotaxis (e.g., cells expressing BLT1)to chemotaxis inhibition for cells expressing CXCR4 can be at leastabout 1.5, at least about 2.0, at least about 3.0, at least about 5.0,at least about 10, at least about 15, or at least about 20.

In some embodiments, for a compound of Formula (I), the IC50 relating tothe corresponding chemotaxis index of a CXCR4-expressing cell can be nomore than about 50 μM, no more than about 35 μM, no more than about 25μM, no more than about 15 μM, no more than about 10 μM, or no more thanabout 5 μM. Some compounds of Formula (I) can have selectivity for CXCR4chemotaxis inhibition, as demonstrated by the relative IC50s. Forexample, the ratio of IC50 chemotaxis inhibition for cells expressingnon-CXCR4 inducing chemotaxis (e.g., cells expressing BLT1) to the IC50for chemotaxis inhibition for cells expressing CXCR4 can be at leastabout 1.5, at least about 2.0, at least about 3.0, at least about 5.0,or at least about 10.

In some embodiments, the compounds of Formula (I) can provide areduction in the intracellular calcium mobilization of a CXCR4expressing cell, measured using normalized Δ fluorescence relative toCXCL12 of no more than about 0.5, no more than about 0.3, no more thanabout 0.2, or no more than about 0.1. In some embodiments, for acompound of Formula (I), the IC50 for the corresponding intracellularcalcium mobilization can be no more than about 50 μM, no more than about40 μM, no more than about 25 μM, no more than about 10 μM, or no morethan about 5 μM.

Some embodiments of the invention include administration of at least onecompound of Formula (I) to a cell. The cell can be a unicellularorganism, or can be obtained from a multicellular organism, e.g.,isolated cells from a multicellular host. The cell can be one of manycells, treated. The cell can include eukaryotics and prokaryotics, suchas strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi,yeast, insect cells such as Spodoptera frugiperda (SF9), animal cellssuch as CHO and mouse cells (e.g., Lewis lung carcinoma cells, B16F10melanoma cells, and TC-1 cervical carcinoma cells), African green monkeycells (such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10), and human cells(e.g., HS27 cells, MCF7 cells, MDA-MB-231 cells, A549 cells, THP-1cells, and 300.19 cells), as well as plant cells. Of course, the cellmay be transfected with one or more genes.

The compounds of Formula (I) can be administered to animals by anynumber of administration routes or formulations. The compounds ofFormula (I) can also be used to treat animals for a variety of diseases.Animals include but are not limited to canine, bovine, porcine, avian,mammalian, and human.

Diseases that can be treated using the compounds of Formula (I) include,but are not limited to cancers (such as cancerous tumors), systemiclupus erythematosus, HIV, Epstein barr virus, coronary artery disease intype II diabetes melitus, chronic rhinositis, carotid artery stenosis,choroidal neovascularization, bladder hyperreflexia, and nephrosclerosis(also referred to as hypertensive nephropathy). Cancers that can betreated include, but are not limited to, basal cell carcinoma, thyroidcancer, squamous cell carcinoma, neuroblastoma, ovarian cancer,melanoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer,colon cancer, lung cancer, pancreatic cancer, prostate cancers, chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia,rhabdomyosarcoma, Glioblastoma multiforme, meningioma, bladder cancer,gastric cancer, Glioma, oral cancer, nasopharyngeal carcinoma, kidneycancer, rectal cancer, stomach cancer, uterine cancer, and leukemias.

Treatment can include that of angiogenesis and metastasis in severaltumor types, including, but not limited to, basal cell carcinoma,thyroid cancer, squamous cell carcinoma, neuroblastoma, ovarian,melanoma, renal cell carcinoma, hepatocellular carcinoma, breast, colon,lung, pancreatic, and prostate cancers. In some instances, treatment canresult in the reduction of tumor size, the reduction in the number oftumors, or both.

In some embodiments, diseases that can be treated include cancers of thelung, lymph node, bone marrow, and liver tissues. In still otherembodiments, treatments include but are not limited to, (1) cancers thathave invasive responses by modulation of actin modification (e.g., incells expressing CXCR4), (2) cancers that have invasive responses bymodulation of pseudopodia formation (e.g., in cells expressing CXCR4),(3) tumor cells by modulating tumor cell migration (e.g., to sites wherenon-malignant stromal cells express CXCL12), and (4) tumor cells bymodulating homing of the neoplastic cells (e.g., to sites wherenon-malignant stromal cells express CXCL12). Other cancers treatmentscan include treatments that interfere with CXCR4, for example, byinhibiting chemotaxis or by inhibiting intracellular calciummobilization. In some embodiments, the compounds of Formula (I) caninhibit CXCR4-mediated chemotaxis and signaling, for example byintracellular calcium mobilization. Sometimes this can result ininhibitory effects of the compounds of Formula (I) that are maintainedin the face of increasing production of CXCL12 by cells in the tumormicro-environment.

Treatment can also include one or more of surgical intervention,chemotherapy, radiation therapy, hormone therapies, immunotherapy, andadjuvant systematic therapies. Adjuvants may include but are not limitedto chemotherapy, radiation therapy, and hormone therapies, such asadministration of LHRH agonists; antiestrogens, such as tamoxifen;high-dose progestogens; aromatase inhibitors; and/or adrenalectomy.Chemotherapy can be used as a single-agent or as a combination withknown or new therapies.

In some embodiments, the administration of at least one compound ofFormula (I) is an adjuvant cancer therapy or part of an adjuvant cancertherapy. Adjuvant treatments include treatments by the mechanismsdisclosed herein and of cancers as disclosed herein, including, but notlimited to metastasis and tumors. Corresponding primary therapies caninclude, but are not limited to, surgery, chemotherapy, or radiationtherapy. In some instances, the adjuvant treatment can be a combinationof chemokine receptor antagonists with traditional chemotoxic agents orwith immunotherapy that increases the specificity of treatment to thecancer and potentially limits additional systemic side effects. In someinstances, the compound of Formula (I) can act as a CXCR4 inhibitor byblocking the metastatic homing of the cancer cells which may cause deathvia anoikis (e.g., by inducing detachment of anchorage-dependent cellsfrom surrounding extracellular matrix). In still other embodiments,compounds of Formula (I) can be used as adjuvant with otherchemotherapeutic agents. The use of a Formula (I)-based therapy mayreduce the duration of the dose of both drugs and drug combinationsreducing the side effects.

The route of administration of the compounds of Formula (I) may be ofany suitable route such as that which provides a concentration in theblood corresponding to a therapeutic concentration. Administrationroutes that can be used, but are not limited to the oral route, theparenteral route, the cutaneous route, the nasal route, the rectalroute, the vaginal route and the ocular route. The choice ofadministration route can depend on the compound identity, such as thephysical and chemical properties of the compound, as well as the age andweight of the animal, the particular disease, and the severity of thedisease. Of course, combinations of administration routes can beadministered, as desired.

One or more compounds of Formula (I) can be part of a composition andcan be in an amount (by weight of the total composition) of at leastabout 0.001%, at least about 1%, at least about 10%, at least about 25%,at least about 50%, no more than about 75%, no more than about 90%, nomore than about 95%, no more than about 99% or no more than about99.99%.

One or more compounds of Formula (I) can purified or isolated in anamount (by weight of the total composition) of at least about 0.001%, atleast about 1%, at least about 10%, at least about 25%, at least about50%, no more than about 75%, no more than about 90%, no more than about95%, no more than about 99%, no more than about 99.99%, from about 1% toabout 95%, from about 10% to about 90%, or from about 25% to about 75%.

In some embodiments, one or more compounds of Formula (I) can be used aspart of a pharmaceutical composition. “Pharmaceutical composition” meansa composition suitable for use in the treatment of animals. In someinstances, the pharmaceutical composition is non-toxic and does notcause additional side effects compared to the drug delivered. In sometherapies which are toxic (e.g., some cancer therapies), apharmaceutical composition can deliver an amount of drug (e.g., one ormore of compounds from Formula (I)) sufficient to kill or alter thediseased cells (e.g., cancer cells or tumor cells) and not kill (oralter to a lesser extent) the non-diseased cells; there may be sideeffects inherent to the drug (e.g., the drug may harm the patient or thedrug may be toxic or harmful to some non-diseased cells in the patient).

“Therapeutically effective amount” means an amount effective to achievea desired and/or beneficial effect. An effective amount can beadministered in one or more administrations. For some purposes of thisinvention, a therapeutically effective amount is an amount appropriateto treat an indication such as cancer. By treating an indication ismeant achieving any desirable effect, such as one or more of palliate,ameliorate, stabilize, reverse, slow, or delay disease progression,increase the quality of life, or to prolong life. Such achievement canbe measured by any method known in the art, such as physical measurementof tumor size, number of tumors, cell chemotaxis, cell migration, cellcalcium release, cell phenotype, monitoring of the level of cancerousantigens in blood serum, or measuring patient life.

One or more compounds of Formula (I) can be part of a pharmaceuticalcomposition and can be in an amount of at least about 0.001%, at leastabout 1%, at least about 10%, at least about 25%, at least about 50%, nomore than about 75%, no more than about 90%, no more than about 95%, nomore than about 99%, no more than about 99.99%, from about 1% to about95%, from about 10% to about 90%, or from about 25% to about 75%. Thepharmaceutical composition can be presented in a dosage form which issuitable for the oral, parenteral, rectal, cutaneous, nasal, vaginal, orocular administration route. The pharmaceutical composition can be ofthe form of, for example, tablets, capsules, pills, powders granulates,suspensions, emulsions, solutions, gels (including hydrogels), pastes,ointments, creams, plasters, drenches, delivery devices, suppositories,enemas, injectables, implants, sprays, aerosols or other suitable forms.

In some embodiments, the pharmaceutical composition can include one ormore formulary ingredients. A “formulary ingredient” can be any suitableingredient (e.g., suitable for the drug(s), for the dosage of thedrug(s), for the timing of release of the drugs(s), for the disease, forthe disease state, or for the delivery route) including, but not limitedto, water (e.g., boiled water, distilled water, filtered water,pyrogen-free water, or water with chloroform), sugar (e.g., sucrose,glucose, mannitol, sorbitol, xylitol, or syrups made therefrom),ethanol, glycerol, glycols (e.g., propylene glycol), acetone, ethers,DMSO, surfactants (e.g., anionic surfactants, cationic surfactants,zwitterionic surfactants, or nonionic surfactants (e.g., polysorbates)),oils (e.g., animal oils, plant oils (e.g., coconut oil or arachis oil),or mineral oils), oil derivatives (e.g., ethyl oleate, glycerylmonostearate, or hydrogenated glycerides), excipients, preservatives(e.g., cysteine, methionine, antioxidants (e.g., vitamins (e.g., A, E,or C), selenium, retinyl palmitate, sodium citrate, citric acid,chloroform, or parabens, (e.g., methyl paraben or propyl paraben)), orcombinations thereof.

Pharmaceutical compositions can be formulated to release the activecompound substantially immediately upon the administration or anysubstantially predetermined time or time after administration. Suchformulations can include, for example, controlled release formulationssuch as various controlled release compositions and coatings.

Other formulations (e.g., formulations of a pharmaceutical composition)include those incorporating the drug (or control release formulation)into food, food stuffs, feed, or drink.

The compounds of Formula (I) can be in the form of salts, optical andgeometric isomers, and salts of isomers. Also, the compounds can be invarious forms, such as uncharged molecules, components of molecularcomplexes, or non-irritating pharmacologically acceptable salts, e.g.the hydrochloride, hydrobromide, sulphate, phosphate, nitrate, borate,acetate, maleate, tartrate, salicylate, etc. For acidic compounds, saltsinclude metals, amines, or organic cations (e.g. quaternary ammonium).Furthermore, simple derivatives of the compounds (such as ethers,esters, amides, etc.) which have desirable retention and releasecharacteristics but which are easily hydrolyzed by body pH, enzymes,etc., can be employed.

The methods of treating an organism will involve treatment with anamount of the compound of Formula (I) that is effective to treat thedisease, condition, or disorder that the organism has, or is suspectedof having, or to bring about a desired physiological effect. In someembodiments, the amount of one of at least one compound of Formula (I)is administered to mammals (e.g., humans) at a concentration of about0.05 to about 15 mg/kg body weight, about 0.2 to about 10 mg/kg bodyweight, about 0.5 to about 7 mg/kg body weight, about 0.5 mg/kg, about 1mg/kg, about 3 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg,about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10mg/kg, about 12 mg/kg or about 15 mg/kg. In regard to some conditions,the dosage will be about 6.5 mg/kg human body weight. In some instances,a mouse can be administered a dosage of, about 1 mg/kg, about 5 mg/kg,about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50mg/kg, about 80 mg/kg, about 100 mg/kg, or about 150 mg/kg. Of course,those skilled in the art will appreciate that it is possible to employmany concentrations in the methods of the present invention, and using,in part, the guidance provided herein, will be able to adjust and testany number of concentrations in order to find one that achieves thedesired result in a given circumstance. In other embodiments, thecompounds of Formula (I) can be administered in combination with one ormore other therapeutic agents for a given disease, condition, ordisorder.

EXAMPLES

Methods

Chemotaxis Assay Method: The chemotaxis assay used 300.19 cells whichstably expressed hCXCR4-GFP, as described previously in Pello et al.(Pello et al., Ligand stabilization of CXCR4/delta-opioid receptorheterodimers reveals a mechanism for immune response regulation, Eur. J.Immunol. (2008), Vol. 38, pp. 537-49). Cells were suspended inchemotaxis buffer (RPMI 1640, 1% fetal bovine serum, 1 mM HEPES) at aconcentration of 1×10⁷ cells per ml. Cell chemotaxis was determined byexamining the migration of the 300.19 cells in response toadministration of 50 μM test compound plus an optimized concentration ofCXCL12 using a chemotaxis assay that was previously described in Basu etal. (Basu et al., Critical role for polar residues in couplingleukotriene B4 binding to signal transduction in BLT1, J. Biol. Chem.(2007) Vol. 282, pp. 10005-17). 100 μL of 300.19 cells from the abovecell suspension were placed in the upper chamber of a Transwell support(Corning Costar, Cambridge, Mass.) and were separated by a 5 micronfilter from a bottom chamber containing 50 μM test compound plus theoptimized concentration of CXCL12 in a final bottom chamber volume of600 μl. After 3 h of incubation at 37° C. in 5% CO₂, the upper chamberwas removed and cells in the lower chamber were counted in a Bürkerchamber. Cell counts for all samples were normalized to a CXCL12 plusDMSO control and results are reported using this normalized measure,referred to herein as the chemotaxis index. In some instances, the IC50concentration (i.e., the concentration at which the chemotaxis index isreduced to 50% of the chemotaxis index of the CXCL12 plus DMSO control)of the test compounds was determined. All assays were performed aminimum of four times; error bars represent the standard deviation ofthe mean.

Intracellular Calcium Mobilization Assay Method: The intracellularcalcium mobilization assay used 300.19 cells which stably expressedhCXCR4-GFP, as described previously in Pello et al. (2008). Cells wereloaded with INDO-I and then pretreated with the 50 μM of the testcompound for 60 seconds prior to stimulation of intracellular calciummobilization with an optimized concentration of CXCL12. The cells had aconsistent peak fluorescence response when treated with CXCL12-only;excitation was at 305 nm and emission was monitored at 405 nm and 490nm; the fluorescence ratio was determined as the ratio of the emissionpeaks at 405 nm and 490 nm. The value Δ florescence is the differencebetween (a) the fluorescence ratio from cells before exposure to CXCL12at its optimized concentration and (b) the fluorescence ratio from cellsfollowing stimulation with the optimized concentration of CXCL12.Decreases in Δ florescence correspond to decreases in intracellularcalcium mobilization, and therefore, decreased signaling. As CXCL12concentration was optimized for each test compound, results arepresented as normalized Δ florescence which is Δ fluorescence divided bythe fluorescence ratio of optimized CXCL12-only stimulation. In someinstances, the IC50 concentration (concentration at which intracellularcalcium mobilization is reduced to 50% of the Δ fluorescence of theCXCL12 plus DMSO control) of the test compounds was determined. Allassays were performed a minimum of four times; error bars represent thestandard deviation of the mean.

Cytotoxicity Assay Method: To determine the potential for test compoundcytotoxicity to influence cell intracellular calcium mobilization andchemotaxis, cell cytotoxicity was assessed using propidium iodide (PI)based flow cytometry. Propidium iodine is impermeable to intact cellmembranes and thus its lack of intercalation into DNA (which increasesPI fluorescence) is a positive indicator of cell viability. Cytotoxicityvalues are presented as percentage of cells staining positive for PI.Lower values of fluorescence indicate less cytotoxicity. All assays wereperformed a minimum of four times; error bars represent the standarddeviation of the mean.

Virtual Screening

Virtual screening is a computational technique that can prescreen vastdatabases of small molecule structures against a three-dimensionalstructure to sec which fit, or dock, into the chosen site. This canreduce the actual physical screening for lead compounds many orders ofmagnitude. CXCR4 is a G-protein coupled receptor (GPCR) that exclusivelybinds stromal-derived factor 1 (SDF-1 also known as CXCL12), a CXCchemokine. The GPCR receptors are characterized structurally by sevenmembrane spanning helical domains, an extracellular amino terminus, anda carboxy terminus on the intracellular side of the membrane. The seventransmembrane (TM) domains are joined by three extracellular (ECL) andthree intracellular (ICL) loops. Homology models were used.

CXCR4 signaling occurs primarily via the pertussis toxin-sensitive Gipathway, composed of Gαi, Gβ, and Gγ. CXCL12 interaction with CXCR4induces dissociation of the Gαi from the trimeric G-proteins, and Gβγactivates several phosphorylation pathways, including ERK1/2, JNK, MAPK,and GSK 3α/β. Activation of the Gβγ subunit also results in signaling bythe Phospholipase C pathway to enhance calcium mobilization as well assignaling by the Phosphoinositide-3-kinase pathway to activate AKT.CXCR4 binding of CXCL12 also serves to augment intracellular cAMP levelsand activate cAMP-dependant signaling pathways, such as PKa or CREB. Thecombined activation of these two factors results in cell migration.Activation of these signaling pathways allows CXCR4 to induce severalchanges in the cell phenotype. CXCR4 enhances cell adhesion by enhancingbinding between integrin couples such as VLA-4 and VCAM-1. CXCR4stimulation produces a phenotype with enhanced invasiveness byincreasing AKT mediated MMP9 expression. Finally, CXCR4 facilitatescytoskeletal rearrangement by stimulating actin polymerization byformation of F-actin.

A fully solvated lipid bilayer 25 nanosecond molecular dynamicssimulation of CXCR4 coupled to the G-proteins was built. This causedmovement of CXCR4 and provided four areas to target, the CXCR4extracellular pocket, the CXCR4 intracellular loops, and thecomplementary site on Gαi.

Compounds Identified by Virtual Screening of the Extracellular Domain

Some compounds were identified by virtual screening (3,300,000 ZINCcompound library using DOCK and FlexX) against our reported T140 bindingsite (an extracellular domain of CXCR4) of the active form of CXCR4(N119A). See, Trent et al., Lipid bilayer simulations of CXCR4 withinverse agonists and weak partial agonists, J. Biol. Chem. (2003) Vol.278, pp. 47136-44. In addition to the native sequence, three mutants ofCXCR4 residue N119 were generated and tested. Two residues were in aninactive conformation (Native and N119K) and two residues were in anactive conformation (N119A and N119S). Compounds I-38 to I-46 wereidentified using the active conformation of CXCR4 in this virtualscreen.

Experiments on Compounds Identified by Virtual Screening of theExtracellular Domain

FIG. 1A shows the effect of the identified compounds on CXCL12-mediatedchemotaxis. Data are presented as a percentage of THP-1 cells(obtainable from ATCC accession number TIB-202) migrating toward aCXCL12 gradient of 500 ng/ml, normalized to the control, which was theCXCL12 only treatment. All assays were performed a minimum of four timeswith inhibitor concentration of 10 μM, except for I-61 and I-62 whichused an inhibitor concentration of 1 μM. Error bars represent thestandard deviation of the mean.

FIG. 1B shows the selectivity of chemokine receptors as determined bycompetition by other chemokines (i.e., RANTES, a CCR5 agonist).Chemotaxis assays were performed with selected compounds at aconcentration of 10 μM. Chemokines signaling via CXCR4 (CXCL12 atconcentration of 500 ng/ml) and CCR5 (RANTES at concentration of 300ng/ml) were utilized to assess the receptor specificity of eachcompound. Each pair of columns for a lane corresponds to two separaterepresentative experiments. The negative control samples (lanes 2 and 7)correspond to the chemotaxis without CXCL12 or RANTES. The CXCL12 andRANTES lanes are positive controls corresponding to CXCL12 alone (lanes1 and 6) or RANTES alone (lanes 4 and 9) mediated chemotaxis. Theexperimental samples (lanes 3, 5, 8, and 10) correspond to CXCL12- orRANTES-mediated chemotaxis when the cells are co-incubated with I-38 orI-46. I-38 was found to specifically inhibit CXCR4 mediated migration ofTHP-1 cells, while I-46 was found to inhibit migration to bothchemokines.

FIG. 1C shows inverse agonist activity against CXCR4 of 1-38 and 1-42 to1-58. A constitutively active mutant of CXCR4 coupled to a FUS 1-lacZreporter gene was expressed in yeast S. cervisiae strain CY12946 andutilized to assess inverse agonist activity. In this assay, CXCR4activity was functionally coupled to the pheromone response pathway,which was measured by the activity of a Fusion-LacZ reporter gene.Stimulation of CXCR4 resulted in a proportional increase in activity ofthe LacZ reporter, which increased the galactosidase activity of theyeast cell. When these cells were exposed to a fluorescent substrate, inthis case fluorescin-di-β-D-galactopyranoside, the galactosidaseconverted the substrate into a fluorescent substance. Increased CXCR4activity, such as that induced by an inverse agonist, produced increasedgalactosidase activity, which in turn increased fluorescence. SeePhillips et al., The stromal derived factor-1/CXCL12-CXC chemokinereceptor 4 biological axis in non-small cell lung cancer metastases. AmJ Respir Crit Care Med, 2003. 176(12): p. 1676-86.

All compounds were tested at a concentration of 10 μM. The negativecontrol with buffer addition only, demonstrates the basal level ofsignaling activity. The y-axis of FIG. 1C is theβ-galactosidase activityof the samples normalized to buffer alone. FC131, a cyclic pentapeptideinhibitor of CXCR4 showing inverse agonist activity (i.e., repression oflacZ expression) was used as a positive control. Error bars indicate thestandard deviation. All compounds showing inhibitory activity in thechemotaxis assays were then tested for toxicity.

Some experiments investigating an in vitro metastasis model forchemotaxis were directed at testing the responsiveness of the derivedhCXCR4-GFP expressing 300.19 cells to AM3100, an established inhibitorof CXCR4. These experiments also established controls for DMSO, themedium in which the inhibitors were suspended. Using the chemotaxisassay method described above, hCXCR4-GFP expressing 300.19 cellchemotaxis was completely inhibited by treatment with AMD3100, atconcentrations ranging from 50 nM to 100 nM (data not shown). There waslittle chemotaxis in response to DMSO alone and no significantdifference between CXCL12 treated cells and those treated with CXCL12plus DMSO (data not shown).

Compounds were investigated for their potential to inhibit chemotaxis ofhCXCR4-GFP expressing 300.19 cells. FIG. 2A show results from thechemotaxis assay method using of nine compounds at 50 μM. Two compounds,I-38 (0.21) and I-43 (0.064) inhibited chemotaxis. FIG. 2B shows thatI-38 had a chemotaxis IC50 of 5 μM, while I-43 had a chemotaxis IC50 of20 μM.

FIG. 3A shows the effect of compounds on intracellular signaling andcalcium mobilization using the intracellular calcium mobilization assaymethod described above. I-39 (0.31), I-44 (0.29), and I-46 (0.067)inhibited intracellular calcium mobilization to a statisticallysignificant greater extent than AMD3100 (0.51). FIG. 3B shows IC50 ofintracellular calcium mobilization for I-39, I-40, I-42, I-44, and I-46.I-38 displays significant reduction of chemotaxis, but does notsignificantly inhibit intracellular calcium mobilization.

FIG. 4 shows cytotoxicity of compounds using the cytotoxocity assaymethod described above, using a test compound concentration of 50 μM.Compounds I-40 (88.3%) and I-44 (99.8%) both displayed increases incytotoxicity compared to untreated (3.54%) and DMSO (3.65%) treatedcontrols. Compound I-38 had a value of 7.93%.

Intracellular Loop Virtual Screening (ILVS)

The modeled structure of the CXCR4-Gαi was used to identify residues onthe interaction surfaces of the two proteins. The CXCR4 residuesidentified were all located on the three intracellular loops and theC-terminal tail; these regions have been identified as relevant forCXCR4 signal transduction. The Gαi residues were all within an eightangstrom proximity of CXCR4. These residues were used in the virtualdocking program Surflex. See, Jain, A. N., Surflex: fully automaticflexible molecular docking using a molecular similarity-based searchengine, J. Med. Chem. (2003) Vol. 46, pp. 499-511. Surflex was used todesign residue-based protomols, virtual docking templates used bySurflex, for the CXCR4 interaction surface and the Gαi surface. The ILVSwas set up using the ZINC compound library comprising 2.5 millioncompound structures. These were individually docked, scored, and rankedby Surflex using the GRID distributed computing system. The top 500compounds were re-submitted as a separate subset using higher accuracydocking parameters in Surflex and the final list of the top thirty sevencompounds were selected for assays using human CXCR4 cell lines.Compounds I-1 to I-37 were identified using this virtual screen.

Experiments on Compounds Identified by Virtual Screening of theIntracellular Domains

FIG. 5 a shows chemotaxis inhibition by some compounds using thechemotaxis assay method described above. Experiments with thirty sevencompounds at 50 μM identified the twenty three compounds in FIG. 5Awhich significantly inhibited chemotaxis. Compounds I-1, I-14, I-15,I-19, I-20, I-28, I-33, I-34, and I-35 produced chemotaxis index valuesof less than 1, but were not significantly less than half of the controlvalue. Compounds I-2, I-3, I-10, I-23, and I-30 produced chemotaxisindex values of greater than 1. FIG. 5B shows dose response analysisperformed on the 23 active compounds which had significant reductions ofchemotaxis below 50% of control, revealing that nine of the twenty threeactive compounds had chemotaxis IC50 values of 10 μM or less. I-36 has achemotaxis IC50 of 1 μM.

I-1 to I-37 were screened at 50 μM for inhibition of intracellularcalcium mobilization, using the intracellular calcium mobilization assaymethod described above. FIG. 6A shows that treatment with eleven of thethirty seven compounds resulted in significant reductions inintracellular calcium mobilization. Compounds I-3, I-6, I-7, I-8, I-10,I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-31, I-32, I-34, I-35, andI-37 all produced normalized Δ fluorescence values of less than 1, butwere not significantly less than half of the control value. CompoundsI-1, I-11, I-14, I-17, I-18, I-26, I-29, I-30, and I-33 all produced Δfluorescence values of greater than 1. FIG. 6B shows dose response offourteen compounds. I-9 (1 μM), I-12 (5 μM), I-13 (5 μM), I-25 (1 μM),and I-28 (10 μM) had intracellular calcium mobilization IC50 values ofless than 10 μM.

Cell cytotoxicity was assessed using propidium iodide (PI) based flowcytometry, as described in the cytotoxicity assay method describedabove, using a test compound concentration of 50 μM. FIG. 7A shows thatcompounds I-2 (63.5%), I-4 (98.8%), I-7 (53.4%), I-9 (81.2%), I-16(67.9%), I-25 (23.4%), and I-36 (21.0%) all displayed cytotoxicity abovethat of untreated (U) (14.5%) and DMSO-treated (14.6%) controls.

To further investigate the cytotoxicity of these compounds, doseresponse assays were conducted at concentrations of 50 μM, 10 μM, 5 μM,and 1 μM to determine the IC50, as shown in FIG. 7B. Cell cytotoxicitywas assessed using the cytotoxicity assay method described above, withvarying concentrations of test compound. None of the compounds showedcytotoxicity greater than that of the control samples at 1 μMconcentrations. FIG. 7B also shows that compounds I-9, I-25, and I-36displayed no cytotoxicity beyond the initial 50 μM screeningconcentration. Compounds I-2, I-7, and I-16 all retained cytotoxicity at10 μM. Only compound I-4 maintained cytotoxicity at 5 μM.

To examine specificity of some of the compounds for CXCR4, theiractivity was tested against a non-family member class A Gprotein-coupled receptor, BLT1. Using the chemotaxis assay method, FIG.8A shows that compounds can inhibit chemotaxis in cells expressing CXCR4and in cells expressing BLT1. I-38 and I-43 also inhibited LTB4-inducedchemotaxis. I-38 and I-43 inhibited BLT1-mediated chemotaxis (0.23) and(0.045) to about the same extent as CXCR4-mediated chemotaxis (0.21) and(0.064), respectively.

Using the chemotaxis assay method described above, FIG. 8B showsadditional measurements of chemotaxis inhibition in cells expressingCXCR4 and in cells expressing BLT1. There were small differences betweenCXCR4- and BLT1-mediated chemotaxis for compounds I-4, I-5, I-6, I-8,I-16, and I-32. Compound I-25 inhibited CXCR4-mediated chemotaxis(0.030) to a 12-fold greater extent than BLT1-mediated chemotaxis(0.38). Compound I-36 inhibited CXCR4-mediated chemotaxis (0.12) to a3.5-fold greater extent than BLT1-mediated chemotaxis (0.44).

FIG. 9 shows measurements for dose response in LTB4-mediated chemotaxisand in CXCL12-mediated chemotaxis. Some of the compounds had IC50 valuesthat were lower for BLT1 than for CXCR4.

FIG. 10A shows intracellular calcium mobilization in cells expressingCXCR4 and in cells expressing BLT1. At 50 μM test compoundconcentrations in BLT1-expressing cells, I-44 (0.040) and I-46 (0.017)inhibited intracellular calcium mobilization. FIG. 10A also shows thatthese compounds produced reductions in CXCL12-mediated intracellularcalcium mobilization, (I-44 (0.29) and I-46 (0.067)), suggesting thattheir inhibition of calcium flux may not be specific to CXCR4. I-38displayed little reduction of intracellular calcium mobilization.

FIG. 10B shows compound specificity screening of compounds thatinhibited intracellular calcium mobilization in cells expressing CXCR4and in cells expressing BLT1. The intracellular calcium mobilizationassay method described above was used. There was no significantdifference between CXCR4- and BLT1-mediated intracellular calciummobilization for compounds I-2, I-4, I-9, I-12, I-13, and I-28. CompoundI-15 inhibited CXCR4-mediated intracellular calcium mobilization (0.45)to an almost 3-fold greater extent than BLT1-mediated intracellularcalcium mobilization (0.16).

FIG. 11 shows measurements of dose response in CXCR12-mediated and inLTB4-mediated intracellular calcium mobilization. Differences in theIC50 for intracellular calcium mobilization were relatively small forI-2 (40 μM for BLT1 vs. 45 μM for CXCR4), I-4 (40 μM for BLT1 vs. 45 μMfor CXCR4), I-12 (2.5 μM for BLT1 vs. 5 μM for CXCR4), I-13 (2.5 μM forBLT1 vs. 5 μM for CXCR4), and I-16 (30 μM for BLT1 vs. 35 μM for CXCR4).The IC50 of intracellular calcium mobilization was significantly lowerfor CXCR4 than BLT1 for I-9 (1 μM for CXCR4 vs. 20 μM for BLT1), I-25 (1μM for CXCR4 vs. 40 μM for BLT1), and I-28 (10 μM for CXCR4 vs. 22.5 μMfor BLT1). The IC50 of intracellular calcium mobilization was higher forCXCR4 than BLT1 for I-15 (50 μM for CXCR4 vs. 30 μM for BLT1).

Cell Proliferation Assays

To determine the potential for compounds to impact the proliferation ofcells, cell growth was assessed capacity using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. The effect of these compounds was determined for four cell lines:HS27, a “normal” fibroblast cell line; MCF7, an estrogen-dependantnon-metastatic breast cancer cell line; MDA-MB-231, anestrogen-independent metastatic breast cancer cell line; and A549, alung cancer cell line. Cells were left untreated, treated with 12.5 mMDMSO, or treated with varying concentrations of four compounds, (I-9,I-25, I-36, and I-37), then incubated for 4 days, before analysis.Absorbance at 490 nm represents proliferating cells. For samples usingcompounds, these values are normalized to untreated samples.

I-37 did not inhibit proliferation for any tested cell line. In thenormal cell line HS27 cells, FIG. 12A shows both I-25 and I-36 displayedmarginal proliferation inhibition at higher concentrations, but theseeffects abated as concentration dropped below 20 μM. I-9 consistentlyinhibited cell proliferation even at concentrations nearing 1 μM. InMCF7 cells, FIG. 12B shows I-9 inhibited cell proliferation with an IC50value of 5 μM. I-36 had an IC50 value of 12.5 μM. I-25 only marginallyinhibited cell proliferation of MCF7 cells at high concentrations.

In MDA-MB-231 cells, FIG. 12C shows I-9 inhibited cell proliferationwith an IC50 value of 5 μM. I-25 had an IC50 value of 10 μM forMDA-MB-231 cells. I-36 had an IC50 value of 30 μM for MDA-MB-231 cells.Finally, in A549 cells, FIG. 12D shows I-9 inhibited cell proliferationwith an IC50 value of 7.5 μM. I-25 had an IC50 value of 10 μM for A549cells. I-36 had an IC50 value of 22.5 μM for A549 cells.

The three compounds illustrate three different avenues of proliferationinhibition. I-9 is generally inhibitory, affecting both normal andcancer cells at levels lower than the cytotoxicity levels (See FIGS. 7Aand 7B). I-25 inhibited the growth of metastatic cancer cell lines A549and MDA-MB-231, but not normal cells or non-metastatic MCF7 cells.Finally, I-36 inhibited the growth of non-metastatic MCF7 cells,metastatic cancer cell lines to a lesser extent, and did not inhibitproliferation of HS27 cells. These results are intriguing as a CXCR4inhibitor need not be cytotoxic to function, for instance I-36 inhibitschemotaxis but does not affect cell proliferation.

In Vivo Methods and Results

FIG. 13 displays inhibition by some CXCR4 antagonists of metastasis ofTC-1 tumor cells in a syngenic mouse model of lung cancer metastasis.FIG. 13A shows the expression of CXCR4 on TC-1 cells, and FIGS. 13B and13C show that AMD3100 and compounds I-38, I-28, and I-36 reduced miceTC-1 metastatic tumor incidence and burden in lungs compared to theirrespective controls (PBS and Vehicle).

Development of Animal Model for CXCR4 Dependent Cancer Metastasis inLungs: To establish a CXCR4 dependent lung tumor metastasis model, wefirst screened several cancer cell lines that are known to metastasizeto lungs in mice models to determine which cancer cell linesendogenously express CXCR4 receptor. The mice cell lines screened wereLewis lung carcinoma, B16F10 melanoma, and TC-1 cervical carcinoma. Micecell lines were screened using flow cytometry-based surface receptorstaining using a fluorochrome labeled antibody to mouse CXCR4 receptor.Among these cell lines, TC-1 cells expressed the highest levels ofmembrane CXCR4 receptor. TC-1 cells that were injected intravenouslyformed massive metastatic tumors in lungs; in contrast, when these micewere simultaneously treated with the FDA-approved CXCR4 antagonistAMD3100, metastasis of TC-1 was significantly inhibited. The TC-1 cellline was generated by transfection of murine lung squamous epithelialcells by Human pappillomavirus (HPV) oncogenes E6, E7, and activatedRas. The TC-1 cells are also expected to serve as a pulmonary metastaticcancer animal model because a high incidence of lung cancer has beenfound in women with previous HPV-related (human papillomavirus)urogenital and anal neoplasias than in individuals without thisparticular clinical history.

Test for Toxicity: Mice were treated by intra-peritoneal injections ofthree of compounds I-28, I-36, and I-38 at the first dose of 5 mg/kg ofbody weight, followed by a second injection of either 20 mg/kg of bodyweight or 40 mg/kg of body weight two days later. This treatment did notinduce any apparent toxicity as observed by the loss of body weight orsigns of physiological distress (such as, mice looking sick orlethargic), over the period of 3 weeks.

Antagonists (Lead Compounds) Inhibit Cancer Metastasis in TC-1 PulmonaryMetastatic Cancer Mice Model: TC-1 cells (5×10⁵) were injectedintra-venously (i.v.) into the tail vein of C57BL/6 mice. 5 mg/kg bodyweight of AMD3100, I-28, I-36, or I-38 were also injected at the samedose intra-peritoneal (i.p.) every day for 21 days starting from the daybefore the tumor cell injection. Mice were sacrificed and lungs wereperfused and collected on day 21 post tumor challenge. Lungs were storedin formalin for 16 hrs and then transferred into 70% ethanol until thelung lobes became lucid. All five lung lobes were separated. Tumors werecounted using a magnifying glass or a microscope. Tumor sizes weremeasured using a digital caliper. The total number of tumors counted inall five lung lobes of an individual mouse is reported as the tumorincidence. Mean tumor sizes were added together to calculate cumulativetumor size, which is reported as the tumor burden for each mouse. Thedata shown in FIG. 13 demonstrate that all compounds are effectiveinhibitors of lung metastasis. In particular, I-38 reduced themetastatic tumor incidence and burden to the lungs compared to the othertreatments in TC-1 pulmonary metastatic mice model. I-38 was found to bemore effective than AMD3100. I-28 and I-36 appeared to have similarefficacy as AMD3100. The average tumor burden in the I-38 treated miceis reduced by more than 60%.

It is noted that terms like “preferably,” “commonly,” and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

As used in the specification, “a” or “an” may mean one or more. As usedin the claims, when used in conjunction with the word “comprising”, thewords “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more. As used in thespecification, the phrase “e.g.” means “for example, but not limited to”in that the list following “e.g.” provides some examples but is notmeant to be a fully inclusive list.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of theinvention.

What is claimed is:
 1. A method for treating a CXCR4 expressing cancerin an animal comprising administering a composition comprising acompound of Formula (I) to the animal, wherein the CXCR4 expressingcancer is breast cancer, ovarian cancer, colon cancer, melanoma,squamous cell carcinoma, chronic lymphocytic leukemia, rectal cancer,lung cancer, or prostate cancer, and the compound of Formula (I) is


2. The method of claim 1, further comprising identifying an animal withthe CXCR4 expressing cancer.
 3. The method of claim 1, wherein the CXCR4expressing cancer comprises a cancerous tumor.
 4. The method of claim 1,wherein the CXCR4 expressing cancer comprises a cancerous tumor and thetreating results in a decrease in the size of the cancerous tumor. 5.The method of claim 1, wherein the CXCR4 expressing cancer comprises acancerous tumor and the treating results in a decrease in the number ofcancerous tumors.
 6. The method of claim 1, wherein the CXCR4 expressingcancer is metastasized.
 7. The method of claim 1, wherein the CXCR4expressing cancer is metastasized to the breasts, ovaries, colon,rectum, lungs, or prostate.
 8. The method of claim 1, wherein the CXCR4expressing cancer is breast cancer or lung cancer.
 9. The method ofclaim 1, wherein the administering is part of an adjuvant treatment. 10.The method of claim 1, wherein the compound of Formula (I) inhibitschemotaxis.
 11. The method of claim 1, wherein the compound of Formula(I) inhibits intracellular calcium mobilization.
 12. The method of claim1, wherein the treating results in at least one of modulation of tumorcell migration or modulation of homing of the neoplastic cells.
 13. Themethod of claim 1, wherein the compound of Formula (I) modulatesactivity of CXCR4.
 14. The method of claim 1, wherein the animal is amammal.
 15. The method of claim 1, wherein the animal is a human. 16.The method of claim 1, wherein the administering is by an oral route orby a parenteral route.
 17. The method of claim 1, wherein the treatingcomprises treating metastasis of breast cancer, ovarian cancer, coloncancer, melanoma, chronic lymphocytic leukemia, rectal cancer, lungcancer, or prostate cancer.
 18. A method for treating a CXCR4 expressingcancer in an animal comprising administering a composition comprising acompound of Formula (I) to the animal, wherein the CXCR4 expressingcancer is breast cancer, ovarian cancer, colon cancer, melanoma,squamous cell carcinoma, chronic lymphocytic leukemia, rectal cancer,lung cancer, or prostate cancer, and the compound of Formula (I) is


19. The method of claim 18, further comprising identifying an animalwith the CXCR4 expressing cancer.
 20. The method of claim 18, whereinthe CXCR4 expressing cancer comprises a cancerous tumor.
 21. The methodof claim 18, wherein the CXCR4 expressing cancer comprises a canceroustumor and the treating results in a decrease in the size of thecancerous tumor.
 22. The method of claim 18, wherein the CXCR4expressing cancer comprises a cancerous tumor and the treating resultsin a decrease in the number of cancerous tumors.
 23. The method of claim18, wherein the CXCR4 expressing cancer is metastasized.
 24. The methodof claim 18, wherein the CXCR4 expressing cancer is metastasized to thebreasts, ovaries, colon, rectum, lungs, or prostate.
 25. The method ofclaim 18, wherein the CXCR4 expressing cancer is breast cancer or lungcancer.
 26. The method of claim 18, wherein the administering is part ofan adjuvant treatment.
 27. The method of claim 18, wherein the compoundof Formula (I) inhibits chemotaxis.
 28. The method of claim 18, whereinthe compound of Formula (I) inhibits intracellular calcium mobilization.29. The method of claim 18, wherein the treating results in at least oneof modulation of tumor cell migration or modulation of homing of theneoplastic cells.
 30. The method of claim 18, wherein the compound ofFormula (I) modulates activity of CXCR4.
 31. The method of claim 18,wherein the animal is a mammal.
 32. The method of claim 18, wherein theanimal is a human.
 33. The method of claim 18, wherein the administeringis by an oral route or by a parenteral route.
 34. The method of claim18, wherein the treating comprises treating metastasis of breast cancer,ovarian cancer, colon cancer, melanoma, chronic lymphocytic leukemia,rectal cancer, lung cancer, or prostate cancer.